Development of the UValidate platform for the profiling of topically applied chemical agents.

项目摘要 皮肤是抵御外界环境的第一道防线。当皮肤屏障被紫外线破坏时 辐射(UVR)或有毒化学物质，活细胞层，包括表皮角质形成细胞、黑素细胞和真皮 成纤维细胞受到DNA损伤。如果不修复，这种损伤可能会导致光老化和皮肤老化。 致癌。事实上，皮肤癌是迄今为止最常见的恶性肿瘤，占癌症病例的比例更高 在美国和其他国家，比所有其他器官的总和还要多。防晒霜已被广泛用于 取得了一些成功，但较新的化学过滤器存在严重的缺陷，使目前可用的 产品不受欢迎，并可能对人类健康构成风险。当前的战略，检查有效性和 有机紫外线滤清器的安全性需要重新评估。这些新策略应该会测试UVR的生物学在 防晒霜的存在。此外，它们需要是高吞吐量(HTP)，以便检查 大多数制剂中存在的不同化学阻滞剂在培养细胞中起作用以便于筛选， 并且爱护动物。在第一阶段，我们展示了一种新型UVR HTP筛查平台的原理证明 使用来自单一捐赠者的同基因角质形成细胞和黑素细胞的混合群体， 暴露于防晒活性成分的两种化合物组合中，存在和不存在短时 波(UVB；285-320)和长波太阳能UVR(UVA1；340-400 nm)。这些滤网测量的是活性氧 物种(ROS)、DNA损伤(CPD)和微核，并显示了太阳紫外线之间复杂的相互作用 波长、防晒剂和细胞类型。相比之下，这突显了对我们平台的迫切需求 到目前使用的只测量光谱阻挡的紫外线数量的检测方法。在建议的 第二阶段，我们计划测试化妆品和护肤品市场的已知化学紫外线阻滞剂作为合同 服务，使用一组来自捐赠者的等基因角质形成细胞、黑素细胞和成纤维细胞。在完成时 第二阶段，如果获得资金，UValify将作为一项特别适合处于 寻求优化配方的药物发现、监管机构和老牌制药公司。这方面的进展 建议包括对等基因患者细胞系进行荧光标记，建立2D和3D细胞培养以确定 个体对太阳紫外线照射的反应，利用试剂和分析方法快速确定 DNA损伤及其修复，并设计人工智能软件，以更准确地确定损伤。控制单元， 使用CRISPR技术生成的，将复制DNA修复受损的皮肤细胞和3D皮肤等价物 也将包括在内，以确保实验的严密性和可重复性。目前的危机是由于缺乏非 有毒防晒霜已导致集体诉讼，全球范围内越来越多的人禁止使用防晒霜，以及 从市场上召回配方。因此，这项技术是及时的，需要极大的便利 解决发现和测试安全有效的紫外线阻滞剂的迫切需要，以防止大多数 我们面临的常见的、往往使人虚弱和致命的癌症。

Project Summary Skin is the first line of defense against the outside environment. When the skin barrier is breached by UV irradiation (UVR) or toxic chemicals, living cell layers including epidermal keratinocytes, melanocytes and dermal fibroblasts are subjected to DNA damage. If unrepaired, this damage can lead to photoaging and cutaneous carcinogenesis. In fact, skin cancer is by far the most common malignancy, accounting for more cases of cancer in the US and other countries than all other organs combined. Sunscreens have been used extensively with some success, but newer chemical filters suffer from serious shortcomings that make currently available products undesirable, and can be a risk to human health. Current strategies that examine the effectiveness and safety of organic UV filters need to be reassessed. These new strategies should test the biology of UVR in the presence of sunscreens. Further, they need to be high-throughput (HTP) in order to examine combinations of different chemical blockers that are present in most formulations, work in cultured cells for ease of screening, and be animal-friendly. In Phase I we showed proof-of-principle for a novel UVR HTP screening platform called “UValidate, employing mixed populations of isogenic keratinocytes and melanocytes derived from a single donor, exposed to two-compound combinations of sunscreen active ingredients in the presence and absence of short wave (UVB; 285-320), and long wave solar UVR (UVA1; 340-400 nm). These screens measure reactive oxygen species (ROS), DNA damage (CPDs), and micronuclei and show a complex interplay between solar UV wavelength, sunscreen blockers, and cell types. This highlights the pressing need for our platform, compared to currently-utilized assays that measure only the amount of UV that is blocked by spectrometry. In the proposed Phase II, we plan to test known chemical UV blockers for the cosmetic and skin-care markets as a contracting service, using a panel of donor-derived isogenic keratinocytes, melanocytes and fibroblast. At the completion of Phase II, if funded, UValidate will be offered as a service particularly suited for companies at the early stages of drug discovery, regulatory bodies and established pharma seeking to optimize formulation. Advances in this proposal include fluoro-tagging isogenic patient cell lines, establishing 2D and 3D cell cultures to determine individual responses to solar UV irradiation, utilization of reagents and assays to rapidly determine the types of DNA lesions and their repair, and design of AI software to more accurately determine damage. Control cells, generated using CRISPR technology, will reproduce DNA repair-compromised skin cells and 3D skin equivalents will also be included to ensure experimental rigor and reproducibility. The present crisis due to the lack of non- toxic sunscreens has led to class action lawsuits, banning of sunscreens in growing numbers worldwide, and recall of formulations from the market. Therefore, this technology is timely and needed with great expediency to address the urgent need for the discovery and testing of safe and effective UV blockers to prevent the most common, and often debilitating and deadly cancers that we face.